Delicate balance between cellular proliferation, differentiation and apoptosis is required for normal bone tissue composition and function. Disruption of such balance leads to bone neoplasia or degeneration. Our long-term goal is to elucidate the mechanisms of disregulation of apoptosis in bone neoplasms and find novel treatments targeted at modulation of apoptosis in neoplasia. The goal of this proposal is to expand our previous findings on counter-regulation of apoptosis in osteoblasts (OB) by Runx2 and NF-kB that will serve as preliminary data for our future RO1 project. During growth and remodeling of bone tissue, bone cells differentiate from mesenchymal stem cells to early proliferating OBs to mature post-mitotic OBs of which 30 percent become either osteocytes or lining cells while 70 percent are eliminated by apoptosis. OB apoptosis is, therefore an ultimate part of OB life cycle which is regulated by the microenvironment and bone-specific cytokines. Despite the importance of apoptosis in bone homeostasis, its regulation in OBs remains elusive. We have collected new data indicating that: 1) A key OB differentiation factor, Runx2, also plays a central role in regulation of OB apoptosis by inducing pro-apoptotic Bax via direct binding and activation of the bax gene promoter; 2) Apoptosis is increased in more differentiated OBs in concert with the increase in Runx2 and Bax; 3) Induction of Bax by Runx2 is negatively regulated by the NF-kB signaling. Bax promoter contains NF-kB binding elements; and 4) NF-kB activity is increased in proliferating OBs and in osteosarcoma cells and plays a role in suppression of apoptosis. In addition, others have found that in Bax KO mice, bone volumes are significantly increased in aging mice implicating Bax in regulation of OB function. In this proposal we will determine the specific role of the Runx2/Bax axis in regulation of apoptosis in OBs and get an insight on the role of NF-kB as a modulator of the Runx2/Bax apoptotic axis. Based on our preliminary results and the literature we hypothesize that apoptosis in osteoblasts is counter-regulated by Runx2 and NF-kB which correspondingly induce or repress transcription of pro-apoptotic Bax. To prove our hypothesis we will address the two Specific Aims: 1) To confirm the inhibitory role of NF-kB in regulation of Bax transcription; and 2) To elucidate the effect of interaction of the Runx2/Bax axis with NF-kB on apoptosis sensitivity in osteoblasts and in osteosarcoma cells. This proposal seeks to understand regulatory mechanisms involved in OB apoptosis and how the Runx2/Bax axis and NF-kB interact at the molecular level. This will provide us a rationale for a RO1 project focused on the mechanism of suppression of apoptosis in bone cancer. 1